Project Summary Calcific aortic valve disease (CAVD) affects a large number of people over age 65. Currently, aortic valve replacement surgery is the only available therapy for this disease. Pharmacological intervention of CAVD progression relies on better understanding of the underlying mechanism. Progressive valvular calcification is the main cause of CAVD progression. Elevated levels of bone morphogenetic protein-2 (BMP-2) and transforming growth factor-?1 (TGF-?1) in valvular tissue contribute to the mechanism of progressive aortic valve calcification, and the aortic valve interstitial cells (AVICs) play a critical role in valvular calcification via pro-osteogenic reprogramming. While BMP-2 and TGF-?1 induce AVIC pro- osteogenic reprogramming and myofibroblastic transition, the molecular mechanism underlying the actions of BMP-2 and TGF- ?1 on AVIC is not well understood, and the role of AVIC myofibroblastic transition in valvular calcification is unknown. We found that both BMP-2 and TGF-?1 up-regulate miR- 486 expression and down-regulate miR-204 expression in human AVICs. Further, co-expression of miR- 486 antagomir and 204 mimic suppresses the pro-osteogenic activity in AVICs of diseased human aortic valves, and miR-486 mimic and miR-204 antagomir elevate the levels of osteogenic transcription factors Runx2 and Osx. Further, miR-204 mimic and miR-486 antagomir synergizes in up-regulation of AVIC pro- osteogenic activity. Based on these novel findings, we hypothesize that miR-486 up-regulation and miR- 204 down-regulation promote aortic valve calcification. The major goals of this project are to elucidate the molecular mechanism of aortic valve calcification and to identify potential therapeutic targets for prevention of CAVD progression. To achieve these goals, we will address the following interrelated Specific Aims: 1) to determine the interaction of miR-486 and miR-204 in elevating the pro-osteogenic activity in human aortic valves and AVICs, 2) to test the hypothesis that miR-486 up-regulates human AVIC pro-osteogenic activity through promoting myofibroblastic transition, and 3) to elucidate the mechanism by which TGF-?1 and BMP-2 modulate miR-486 and miR-204 expression in human AVICs. The results of this project will help improve the understanding of the mechanism responsible for CAVD progression and may identify potential targets for pharmacological intervention.